This application seeks to develop substantially improved vectors that can be used in clinical trials for cystic fibrosis (CF) gene therapy. CF is a common, serious genetic disease for which gene therapy is a potential life-saving treatment. CF manifests much of its clinical significance in the lung, which is the tissue of most relevance to CF gene therapy at this time. Clinical trials using adenovirus vectors and liposomes for CF are ongoing, and results indicate that the development of new vectors is needed. Three specific problems will be addressed in this proposal 1) the toxicity and short duration of expression associated with presently utilize vectors in the lung, 2) the inefficiency of vector entry to intact lung epithelium, and 3) the inability to monitor gene transfer in the lung in CF patients without invasive procedures. A new generation of adenovirus vectors, termed delta-vectors, will be used to address these issues, with the idea that these vectors will have potential to be used in the lung of CF patients. Delta-vectors differ from other adenovirus vectors in that they contain no viral coding sequences. These vectors contain the expression cassette of interest, the adenovirus inverted terminal repeats and packaging signal, and any stuffer DNA necessary to bring the DNA molecular to the appropriate size. They are propagated in the presence of a helper virus, which supplies all necessary proteins vector DNA packaging. Recent results using intravenous delivery provide a strong indication that these vectors have great potential for increasing safety and improving duration of expression of therapeutic genes. This proposal will address their potential in the lung by evaluating three specific aims: 1) to compare delta-vectors containing the CF gene to first generation adenoviral vectors for differences in toxicity and duration of gene expression after delivery to the lung; 2) to compare delta-vectors containing the CF gene and modified fiber proteins to those not containing modified fiber proteins for differences in efficiency of viral entry into airway epithelial cells; 3) to evaluate A-vectors containing both a serum reporter gene alpha-fetoprotein (AFP) and CF to determine the utility of AFP as a reporter gene in the lung and the ability to use AFP to follow CF expression, hopefully making the monitoring of gene transfer more effective and less invasive. To acheive these goals, plasmids that can be used to generate a variety of delta-vectors carrying CF and/or AFP expression cassettes will be made by molecular cloning. These plasmids will be used to generate vectors in the presence of helper virus. In addition, helper virus with a fiber gene modified to contain polylysine residues will be made and will be used to propagate the vectors. After vector propagation, the vectors will be tested in the appropriate in vivo and in vitro models to determine their potential value in humans.